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Research Report

Minocycline treatment prevents cavitation in rats after acortical devascularizing lesion

Rui Hua, Wolfgang Walz⁎

Department of Physiology, University of Saskatchewan, 107 Wiggins Road, Saskatoon, Canada SK S7N 5E5

A R T I C L E I N F O

⁎ Corresponding author. Fax: +1 306 966 6532E-mail address: walz@sask.usask.ca (W. W

0006-8993/$ – see front matter © 2006 Elsevidoi:10.1016/j.brainres.2006.03.072

A B S T R A C T

Article history:Accepted 7 March 2006Available online 2 May 2006

Minocycline, a second-generation tetracycline, has been shown to possess neuroprotectiveeffects in animal models of stroke. Pial vessel disruption in adultWistar rats leads to a cone-shaped cortical lesion and turns into a fluid-filled cavity surrounded by a GFAP+ glialimitans 21 days after injury. This mimics the clinical situation in lacunar infarcts.Minocycline was given intraperitoneally at a dose of 45 mg/kg 1 and 12 h after lesioning,followed by 22.5 mg/kg twice daily until 6 days after lesioning. Control rats receivedintraperitoneal injections of equivalent volumes of saline. Cavitation was prevented in fiveout of sixminocycline-treated animals and the glia limitans did not appear as the space wasfilled with GFAP+ reactive astrocytes. However, the number of activated microglia showedno difference between minocycline-treated and -untreated groups. Minocycline did notreduce the number of infiltrating leukocytes, predominately polymorphonuclearneutrophils (PMNs) determined by myeloperoxidase immunoreactivity, or infiltration ofCD3+ lymphocytes. The pial vessel occlusion induced a significant upregulation of IL-1βexpression; however, minocycline treatment did not significantly alter this upregulation ofIL-1β. In this study, we found minocycline facilitated the repopulation of the lesion byreactive astrocytes and therefore prevented cavitation; however, we could not identify themolecular signal.

© 2006 Elsevier B.V. All rights reserved.

Keywords:MinocyclineIschemiaStrokeMicrogliaAstrocytesReactive gliosis

1. Introduction

Minocycline, a second-generation tetracycline, has beenshown to have neuroprotective properties in animal modelsof acute CNS injury and chronic neurological diseases, in-cluding brain ischemia (Yrjanheikki et al., 1998; Yrjanheikki etal., 1999), spinal cord injury (Teng et al., 2004; Lee et al., 2003;Wells et al., 2003), traumatic injury (SanchezMejia et al., 2001),Huntington's disease (Chen et al., 2000), Parkinson disease (Duet al., 2001), and amyotrophic lateral sclerosis (ALS) (Zhu et al.,2002). Most of the evidence suggested the neuroprotectiveeffect of minocycline is associated with inhibiting the inflam-

.alz).

er B.V. All rights reserved

mation process by preventing activation and proliferation ofmicroglia (Tikka et al., 2001) and inhibition of nitric oxide syn-thetase (iNOS) aswell as IL-1β converting enzyme (ICE) expres-sion, thereby preventing IL-1β formation following injury(Yrjanheikki et al., 1998; Du et al., 2001; Chen et al., 2000).Minocycline treatment also reduces expression of the matrixmetalloproteinase's (MMPs), which are involved in the break-down of the blood–brain barrier and subsequent infiltrationof inflammatory cells into the brain (Brundula et al., 2002;Koistinaho et al., 2005). In addition to anti-inflammationeffect,minocycline can also modulate neuronal cell death by a directinteraction with the apoptotic machinery, possibly acting on

.

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caspase-dependent and -independent cell death mechanisms(Chen et al., 2000; Zhu et al., 2002; Wang et al., 2003).

However, these very encouraging results were not found inall studies. Lately, several studies found thatminocycline lacksa beneficial effect or may even have detrimental effects. Thiswas shown for Huntington's disease (Smith et al., 2003; Diguetet al., 2003), Parkinson's disease (Diguet et al., 2004a, Yang etal., 2003), hypotoxic-ischemic brain injury (Tsuji et al., 2004),and spinal cord injury (Zang da and Cheema, 2003). It has evenbeen suggested that many negative effects were never re-ported, and therefore the record of minocycline may be morequestionable than the literature suggests (Diguet et al., 2004b).

With regards to stroke, minocycline showed beneficialeffects in different animal models of global ischemia (Yrjan-heikki et al., 1998; Yrjanheikki et al., 1999), transient andpermanent focal cerebral ischemia (Koistinaho et al., 2005; Xuet al., 2004; Yrjanheikki et al., 1999), and hypoxic-ischemicbrain injury in neonatal rats (Arvin et al., 2002; Yrjanheikki etal., 1999; Fox et al., 2005). However, Tsuji et al. (2004) reportedthat minocycline could exacerbate hypoxic-ischemic braininjury in thedevelopingmousewhileprovidingmildprotectionin rat pups. Fox et al. (2005) found only a transient protection inrat pups. In a permanent middle cerebral artery occlusionmodel (MCAO) on adult mice, minocycline treatments startedbefore MCAO reduced the infarct size, whereas the treatmentstarted after MCAOwas not protective (Koistinaho et al., 2005).Before minocycline can be used in clinical trials with strokepatients, it must be shown to be effective in several types ofstroke models with drug delivery times that are clinicallyrealistic (Diguet et al., 2004b, Yong et al., 2004). In the presentstudy,weuse thepial vesseldisruptionmodel to test theeffectsof minocycline treatment. In this model, rat pial vesseldisruption produces a consistent cone-shaped lesion, whichturns intoa fluid-filled cavity 21daysafter lesioning (WangandWalz, 2003; Wang et al., 2004). Therefore, it mimics conditionsof lacunar infarctions, one of the most frequent human strokepathologies (Arboix and Marti-Vilalta, 2004; Fisher, 1982).Standard minocycline treatment was started an hour afterlesioningand theoutcomewas evaluated basedonhistologicaland immunological parameters.

Fig. 1 – Lesion volume 6 days after lesioning insaline-treated and minocycline-treated rats as measuredwith H&E (n = 6). There is no significant difference (P = 0.63).Data are shown as mean ± SEM.

2. Results

2.1. Minocycline treatment prevents the formation of cysticcavity and glia limitans

A consistent pattern of cortical lesion was demonstrated byH&E staining which corresponded to the disruption area. Oneday after injury, the damaged areawas identified as less densestain and the presence of dying neurons, which lack thestainable nucleic acid. After 3 days, all cells (neurons andastrocytes) in the lesion were gone. Microvessel proliferationand a massive infiltration of blood borne cells were observedin the infarct area. A well-demarcated cortical lesion wasobserved by day 6 and a cystic cavity developed at 21 days (seeFig. 2 of Wang and Walz, 2003).

Because the lesion developed a distinct border by day 6, wechose day 6 as the time point to measure the volume of thecortical lesion from the H&E-stained sections. Minocycline

treatment did not reduce the lesion volume (1.09 ± 0.30 mm3)compared to the saline-treated group (0.91 ± 0.15 mm3) (Fig. 1).However, by day 21, in the minocycline-treated group, only 1out 6 animals had the cavity by day 21 in contrast to 5 out of 6animals that exhibited the cystic cavity in the lesion area inthe control group (Figs. 2A and B).

The cystic cavity is surrounded by a glia limitans (Wangand Walz, 2003). Because minocycline treatment preventedthe cavitation that appears 21 days after injury, weinvestigated whether the rescued tissue is part of thebrain parenchyma and therefore within the tissue enclosedby the glia limitans or outside the glia limitans. GFAPimmunostaining was therefore used as a marker ofastrogliosis. In the saline-treated group, all animals devel-oped a glia limitans on day 21 following injury (Figs. 2Aand C). With minocycline treatment, the glia limitans wasabsent in 5 out of 6 treated rats (Figs. 2B and D) and therescued tissue space was filled with reactive GFAP+astrocytes (Fig. 2D). However, the appearance and distribu-tion of GFAP+ cells in the tissue surrounding the lesion isnot changed by minocycline treatment on days 1, 3, and 6after injury (Fig. 3).

2.2. Minocycline treatment and inflammatory responseafter pial vessel disruption

2.2.1. Microglia activationThere is evidence that the neuroprotective effect of minocy-cline is related to the inhibition of microglial activation(Tikka et al., 2001). To determine whether minocyclineaffects glial cell activation, lectin (GSAB4) histochemistrywas used to evaluation of microglia. As described in theExperimental procedure section, microglial activation afterpial vessel disruption is classified into 3 stages/types: restingmicroglia, activated microglia, and phagocytic microglia/macrophages. The typical morphology of these three types/stages of GSAB4+ microglia in the saline-treated group isshown in Fig. 4.

In the control (saline-treated) group, 1 day after injury,scattered GSAB4-positive small round cells were present inthe affected area, but no resting and activated microgliacould be seen there. In the area surrounding the infarctcore, bushy and highly ramified cells were seen withincreased numbers. The activation and proliferation of

Fig. 2 – Effect of minocycline on cystic cavity and glia limitans 21 days following pial vessel disruption. A cystic cavity andglia limitans developed between days 14 and 21 in saline-treated group (n = 6; panels A and C). With minocycline treatment,the glia limitans was absent and the injured area was filled with reactive GFAP+ astrocytes (n = 6; B and D). (A and B)Representative H&E-stained sections. (C and D) Adjacent sections stained for GFAP. Scale bar: 200 μm.

Fig. 3 – Comparison of GFAP staining in saline-treated (A and C) and minocycline-treated rats (B and D) 6 days after lesioning.(A and B) The overview pictures include cortical lesion and peri-lesion areas. White outline delineates the lesion. (C and D)High magnification of the rectangular crop area in panels A and B. Scale bars: 500 μm (A, B); 50 μm (C, D).

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Fig. 4 – The typicalmorphology of three types/stages of GSAB4+microglia after pial vessel disruption. (A) Restingmicrogliain the cerebral cortex of normal brain, which showed ramifiedmorphology. (B–C) Activatedmicroglia: Morphological changesof activatedmicroglia includeenlargedcell bodiesandcontractionofprocesses.Twomajormorphological typesweredetected inourmodel:highlyramifiedshape (B,enlargedcellbodygivingrise to ramified taperedprocess;day1postlesion;peri-infarct)andabushy shape (C, very large cell bodywith thick, short processes; day 3 postlesion; peri-infarct). (D) Phagocyticmicroglia demonstrating an amoeboid or round brainmacrophage-likemorphology (day 6 postlesion; infarct). Scale bar: 20μm.

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microglia were further increased on day 3. Accumulatedround and amoeboid cells were present within the infarct.A dense zone of bushy microglia mingled with amoeboidcells was observed between the infarct and the surroundingarea thus forming a transitional zone. Surrounding thistransitional zone, most of the microglia were highlyramified. After 6 days, the entire ischemic core was denselycovered by round and amoeboid cells. The transitional zonewas very narrow compared to day 3. Highly ramified cellsmixed with bushy cells were seen immediately surroundingthe infarct core. The density of the GSAB4-positive cells inthe surrounding area is doubled compared to the contra-lateral area, from 14,095 ± 438 to 27,088 ± 4,291 cells/mm3

(P < 0.05; n = 3), indicating a robust activation of microgliaafter pial vessel disruption. In the minocycline-treatedgroup, neither the morphology and distribution of theactivated types nor their densities was changed 1, 3, and6 days following ischemia (Fig. 5). We further quantified thedensity of activated microglia surrounding the infarct coreon day 6 in the minocycline-treated group. Minocycline didnot change the density of GSAB4-positive cells in thecontralateral brain: 13,798 ± 1,621 cells/mm3 in the mino-cycline-treated group and 14,095 ± 438 cells/mm3 in thesaline-treated group. The density of GSAB4-positive cells inthe area surrounding the infarct core (peri-infarct area)showed no significant difference between minocycline-treated (23,610 ± 1,067 cells/mm3) and minocycline-untreated group (27,088 ± 4,291 cells/mm3; Fig. 5G) aswell. These results indicated that minocycline treatmentdid not alter the activation of microglial phenotypes.

2.2.2. Infiltration of leukocytes and T-lymphocytesOnly few Infiltrating leukocytes, predominately polymor-phonuclear neutrophils (PMNs), determined by myeloper-oxidase (MPO) immunoreactivity, were detected in eitherthe contralateral side or the tissue outside the lesion of theipsilateral hemisphere. The MPO immunoreactive cellswere evident in the lesion site at days 1 and 3 after pialvessel disruption (Figs. 6A and B) and only occasionallyseen in the lesion site at day 6. Quantitative analysisshowed that minocycline treatment did not significantlyreduce the invasion of PMNs (Fig. 6C). Only very fewinfiltrating T-lymphocytes (CD3 immunoreactive cells) wereseen around the border of the lesion site on day 1. At day3, their number had increased and reached a peak aroundday 6. The infiltration of CD3 immunoreactive lymphocyteswas not affected by minocycline treatment (data notshown).

2.2.3. IL-1β protein expressionThe amount of IL-1β in the tissue extract was expressed asng/g wet weight of the samples. The pial vessel disruptionresulted in a significant increase in the IL-1β concentrationof injured brain tissue compared to the contralateral side(approximately 1.7-fold, P < 0.05, n = 7). Although minocy-cline treatment showed a trend to reduce the expression ofIL-1β 24 h after treatment, it failed to reach statisticalsignificance (P = 0.12; Fig. 7). We normalized IL-1βconcentration to both brain wet weight and total brainprotein. Both ways of expressing the data showed nosignificant difference of IL-1β level between minocycline-

Fig. 5 – Comparison of GSAB4 staining in the infarct and peri-infarct area of saline-treated (A–C) minocycline-treated rats(D–F) at different time points. (A, D) Day 1; (B, E) day 3; and (C, F) day 6. In the minocycline-treated group, neither themorphological activation of microglia nor density was changed 1, 3, or 6 days following ischemia. White outline delineatesthe lesion. The small picture inset represents high magnification of the rectangular crop area. Scale bar: 300 μm. (G)Quantitative comparison of GSAB4+ cells in the different areas surrounding the lesion on day 6 in saline- andminocycline-treated animals. The inset represents a coronal section with locations of the ischemic lesion and representativefields used in quantification. In both saline- and minocycline-treated groups, the cell density was significantly (P < 0.05)increased in areas immediately surrounding the lesion comparedwith the contralateral area in the same group, but GSAB4+ celldensity showed no significant difference in the minocycline-treated group compared with the saline-treated group.

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treated and -untreated animals, although there was a trendtoward reduction.

3. Discussion

In the present study, we did not find that minocyclinetreatment had an effect on the lesion volume. Therefore, one

has to conclude that there is no neuroprotective effect in thisexperimental irreversible stroke model. The untreated strokelesion in control animals transformed normally into an extra-parenchyma fluid-filled cavity surrounded by the glia limitansas a new barrier. Minocycline treatment rescued this area aspart of the parenchyma as the space was occupied by reactiveastrocytes. Although no neurons were rescued, this actionmay still result in beneficial effects as reactive astrocytes are a

Fig. 6 – Infiltration of neutrophils after pial vessel disruption. (A and B) Photomicrographs ofmyeloperoxidase-immunostainedneutrophils infiltrate in lesion 1 day (A) and 3 days (B) after pial vessel disruption. The white outline indicates the border ofthe infarct zone. (C) Number of polymorphonuclear neutrophils in the lesion at days 1 and 3 after pial vessel disruption.Myeloperoxidase-positive cells were normalizedwith the lesion area. Data are shown asmean ± SD.Minocycline treatment didnot reduce the number of polymorphonuclear neutrophils in the lesion (n = 3). Scale bar: 100 μm.

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major source of trophic factors and other neuroprotectants(Pekny and Nilsson, 2005). We were not able to find themechanism on which the prevention of cavitation is based.Microglial activation and infiltration of leukocytes wereunaffected by minocycline treatment as judged by themorphology and density of the GSAB4-positive cells, CD3 andMPO-positive cells, respectively. Although minocycline treat-

Fig. 7 – ELISA analysis of IL-1β expression in the brain afterpial vessel disruption. The data are expressed in ng/g wettissue weight. Two-way ANOVA was used to analyze thedata. It showed a significant effect of ischemia (P < 0.05), andthe effect of minocycline showed a trend of reduction theexpression of IL-1β but it failed to reach statisticalsignificance (P = 0.12). The data are shown as mean ± SEM(n = 7).

ment showed a trend to reduce the expression of IL-1β, itfailed to reach statistical significance.

So far with regards to stroke-like conditions, minocyclinehas been mainly tested and shown to have neuroprotectiveeffects on the MCAO model, the most common used focalischemic animal model because of its clinical relevance (Xu etal., 2004; Koistinaho et al., 2005; Yrjanheikki et al., 1998;Yrjanheikki et al., 1999). Ischemic injury in the brain resultsin neuronal cell death, with characteristics of both necrosisand apoptosis (Lipton, 1999). Apoptosis, which is temporallyand spatially different from the necrosis in the infarct core,occurs mainly in the surrounding penumbra region (Mer-genthaler et al., 2004). Apoptosis is one of the direct targets ofminocycline action (Domercq and Matute, 2004) and it acts atthemitochondrial level to reduce the apoptosis of neurons andoligodendrocytes in various neurodegenerative disease mod-els (Lee et al., 2003; Zhu et al., 2002; Stirling et al., 2004). Theclass II pial vessel disruption induces a small cortical lesion,whichmimics the irreversible focal ischemia (Wang andWalz,2003). Compared to MCAO, it does not create a sizable penum-bra of sublethal injury in the cortex and striatum (Gotts andChesselet, 2005); thus, there are less neurons subjected todelayed death and subsequently less neurons are a potentialtarget for treatment (Muir and Grosset, 1999).

Another cause for the lack of effect on lesion volume byminocycline treatment could be the time point of the start oftreatment. In our study, the drug was delivered 1 h postinjury.Indeed, in a study on the permanent MCAO, it has been shownthat if minocycline treatment was initiated before lesioning,

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the ischemic infarct was dramatically reduced whereas noeffect was observed when the treatment started 2 h after theonset of the permanent MCAO (Koistinaho et al., 2005). Inanother study of a transient MCAO model on rat pups, mino-cycline treatment starting 2 h after MCA occlusion only re-duced the brain injury at 24 h but not 7 days. They too failed tofind a minocycline induced reduction of microglial activationand of the expression of local proinflammation mediators,such as IL-1β, IL-18, MCP-1, and CINC-1 (Fox et al., 2005).Minocycline did not inhibit microglia activation in the facialnucleus following crush injury either (Fendrick et al., 2005).

We decided to delay treatment by 1 h as this is a morerealistic scenario for a clinical situation compared to pretreat-ment or simultaneous treatment with respect to the time oflesioning. Another factor could be that intraperitoneal deliveryof minocycline may take up to 4 h to peak in the systemiccirculation (Faganet al., 2004). Theminocycline administrationprotocol used in our study was the same as the one used byYrjanheikki et al. (1999) in the MCAO model, and there mino-cycline was successful in rescuing neurons. However, differ-ences in the strokemodels, regional brain areas, experimentalspecies, strains, and other parameters may require differentdosage and protocols of treatment in order to produce signi-ficant neuroprotective effects (Tsuji et al., 2004).

However, despite a lack of effect on the stroke volume byday 6, we found that minocycline abolished the substantialprogressive cavitation,whichdevelops inmore than 80%of thelesioned animals between days 14 and 21 (Wang and Walz,2003). The space occupied by the cavity was occupied inmino-cycline-treated rats by reactive, GFAP+ astrocytes. In addition,the glia limitans, which surrounds the cavity in control ani-mals, was absent in these treated rats. Thus, an importanteffect of minocycline treatment during the first 6 days afterinjury was that the loss of brain parenchyma, which appearedquite later, was prevented. Such an effect is new and has notbeen reported before. Given the importance of lacunar infarc-tions in human stroke pathology, this finding has relevancy inhuman treatment with minocycline. In addition, this effectcould have important ramifications as the space was nowpopulated by reactive astrocytes as part of the parenchyma.There was no apparent barrier separating these reactiveastrocytes from adjacent surviving tissue. As these cells areknown to produce neurotrophic factors, they could thereforehelp salvage neuronal survival, migration, and axonal regen-eration in adjacent areas.

Progressive cavitation is closely related to the inflammatoryresponse (Fitch et al., 1999) and is based on activation ofmicroglia/macrophages and migration of reactive astrocytesout of the affected area. This leads to abandonment byastrocytes and therefore leaves this area more vulnerable toinflammatory damage. Therefore, it may not come as a sur-prise that this process is a natural target forminocycline. In thepial vessel disruption model, we found evidence that shortlyafter injury, the PMNs recruited and infiltrated in the lesionarea followed by delayed infiltration of T cells and activatedmicroglia/macrophages (both hematogenous origin and brainmicroglia) in the ischemic area and boundary zone. Astrocytesabandon the lesion within the first few days, but at the borderthey become reactive and divide. Six days after lesioning, anastrocytic boundary, known as glia limitans, is formed sur-

rounding the lesion. By day 21, the lesion site is transformedinto a fluid-filled cavity (Wang and Walz, 2003; Wang et al.,2004).

In our strokemodel, minocycline treatment did not changeany of the patterns of these cell types in the first 6 days.However, it prevented the formation of the cavity by filling upthe space with reactive astrocytes between days 6 and 21. Itshould be pointed out that the minocycline treatment wasstopped by day 6, when there was not yet a cavity. Reactiveastrocytes must have been migrating back into the lesionedarea and this migration prevented the formation of a fluid-filled cyst. It is usually persistent macrophage/microgliaactivation, which is responsible for the outward migration ofastrocytes first of all (Fitch et al., 1999). Although we did notfind direct evidence for reduced inflammation after minocy-cline treatment, it seems most likely that minocycline ispreventing cavitation through its anti-inflammatory effect onmicroglia early during the first 6 days. Activated microglia/macrophages not only act as scavengers removing tissuedebris, but also release numerous agents that are toxic to CNScells, including inflammatory cytokines and glutamate, whichaggravate secondary tissue injury and modulate the astrocyteresponse by releasing inflammatory cytokines, chemoattrac-tants, such as osteopontin (Wang et al., 1998) and MMPs(Planas et al., 2002). Among all these inflammatory cytokines,interleukin-1β (IL-1β) plays an important role in the signalingbetweenmicroglia and reactive astrocytes (Giulian et al., 1988;Herx and Yong, 2001).

In our study, minocycline did not significantly decrease theproduction of IL-1β 24 h postinjury. Such a lack of effect wasalso reported by Fox et al. (2005). Thus, it is unlikely to be themicroglia–astrocyte signal responsible for the minocycline-induced repopulation by reactive astrocytes. It could very wellbe that this repopulation is not caused by changes in diffusiblesignals but by changes in matrix molecules. Fitch et al. (1999)found that early in the progressive cavitation process, whenthe lesion is abandonedby astrocytes, but still filledwith ahighdensity of microglia/macrophages, there are increased levelsof chondroitin sulfate proteoglycans (CSPGs), which preventedrepopulation of the cavity by astrocytes. Thus, it may well bethatminocycline in the first 6 days is not changing thequantityof microglia activation but specifically downregulates signalswhich prevent astrocyte repopulation, one of which might beCSPG.

4. Experimental procedures

4.1. Surgery

All studies were performed under a protocol approved by theprotocol Review Committee of Animal Care and Supply(#20020024). Male adult Wistar rats (Charles River Inc., StConstant, QC, Canada) 250–350 g were used. After arrival, theanimals were kept for at least 1 week before being used in anyprocedure. Focal cerebral ischemia was induced as describedbyWang andWalz (2003) by disruptingmedium-sized (class II)vessels on the cortical surface of adult rats. Rats were anes-thetizedwith an intraperitoneal injection of ketamine (125mg/kg) and xylazine (7 mg/kg) mixture. A craniotomy was

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performed with a 5-mm-diameter trephine positioned on theright side adjacent to coronal and sagittal sutures. Cool sterilesaline was applied intermittently to prevent overheating fromthe high-speed drilling. After removing the dura, the corticalsurface and the overlying pial vessels were exposed. Medium-sized (class II) pial vessels (Bar, 1980) were disrupted by fine-tipforceps. The piece of bonewas replaced and the scalpwas thenclosedwithwound clip. The body temperature was monitoredthroughout the surgery by a rectal probe and maintained at37 °C by a heating pad. Animals were kept in a cage separatelyunder a warm lamp during the recovery from anesthesia.

4.2. Minocycline treatment

Minocycline (Sigma) was given intraperitoneally at a dose of45 mg/kg 1 and 12 h after lesioning, followed by 22.5 mg/kgtwice daily until 6 days after lesioning (Yrjanheikki et al.,1999). Control rats received intraperitoneal injections of equi-valent volumes of saline.

4.3. Tissue preparation, lectin histochemistry, andimmunohistochemistry

One, 3, 6, or 21 days after lesioning, the rats were anesthetizedwith ketamine (125mg/kg) and xylazine (7mg/kg)mixture andintracardially perfused with PBS followed by formalin–aceticacid–methanol (FAM) 1:1:8. Brains were paraffin-embeddedand cut into 10-μm-thick coronal sections with a microtome.

Microglia were identified bymeans of lectin histochemistrywith the B4-isolectin from Griffonia simplicifolia (GSAB4, Sigma),which specifically binds to alpha-D-galactosyl residues. Itstains all the different stages of microglia (Streit, 1990). Depa-raffinized sections were quenched with 0.3% hydrogen perox-ide and pretreated in citrate buffer (pH 6.0) at 90 °C for about45 min. After blocking in 1% bovine serum albumin, sectionswere incubatedwith biotin-labeledGSAB4 (10mg/ml, Sigma) in1%BSA at 4 °C overnight (Savchenko et al., 2000). Horseradishperoxidase (HRP)-conjugated streptavidin (1ug/ml, Jackson)was applied for 2 h at room temperature. TRITC-conjugatedgoat anti-HRP (1:25, Jackson) was applied for visualizing thebinding site. The nuclei were counterstained with Hoechst33258. We use the following classification for describing thestage of microglial activation (Ziaja and Janeczko, 1999; Katoand Walz, 2000).

1. Resting microglia, which are ramified microglia andpresent in the normal brain. They had the morphology ofround, small cell body with radiating long, thin, branchedprocesses.

2. Activatedmicroglia, which are cells responding to ischemiawith morphological and immunophenotypic changes, butare not phagocytic. Their morphological changes includeenlarged cell bodies and contraction of their processes toshow a more stout morphology. Two major morphologicaltypeswere detected in ourmodel: highly ramified shape, anenlarged cell body giving rise to ramified tapered process; abushy shape, a very large cell body fromwhich issued thick,short processes.

3. Phagocytic microglia, which are full-blown brain macro-phages with an amoeboid or round morphology and

expression of a number of immunomolecules that is sharedwith hematogenous macrophages.

Reactive astrocytes/gliosis were assessed in serial sectionsby direct two-step immunofluorescent staining with antibo-dies to glial fibrillary acidic protein (GFAP). Myeloperoxidase(MPO) and CD3 were used as a marker for polymorphonuclearneutrophils (PMNs) and lymphocyte respectively. Sectionswere incubated with the primary antibody (goat anti-GFAP,1:25, Santa Cruz; rabbit polyclonal antibodies against MPO,1:100, Neo Markers; mouse monoclonal antibody against ratCD3, 1:25, Serotec) at 4 °C overnight, followed by a secondaryantibody conjugated with fluorescence at room temperaturefor 2 h.

Heat-induced antigen retrieval (AR) technique was used inGSAB4 histochemistry and MPO, CD3 immunostaining forretrieval of antigens that have been masked by formalin fixa-tion (Boenisch, 2001). After deparaffinizing and rehydrating thetissue sections, the slides were treated in citrate buffer (pH 6.0)at 90 °C about 45 min and achieved a stronger intensity ofimmunohistochemical staining.

4.4. Lesion volume and cystic cavity

As pointed out in the introduction and elsewhere (Wang andWalz, 2003; Wang et al., 2004), the lesion is filled with cellularmaterial (mostly macrophages and microglia) and has adistinct border by day 6. By day 21, it turns into a fluid-filledcyst with a glia limitans. Lesion volume quantification wasachieved by hematoxylin and eosin (H&E) staining on 6 dayspostsurgery animals. Every tenth section of a series of 10-μm-thick sectionswas stainedwithH&E. The sectionwas capturedby digital camera (Coolsnap fx) mounted on a microscope(Olympus IX71) and the border traced on the image. Thelesioned area on each section was computed from the borderoutline by ImagePro software. The total lesion volume wascalculated by summation of the areas multiplied by the inter-slice distance (100 μm).

The fluid-filled cystic cavity appears between days 14 and21. It is evident from the cell-free area encased by a glialimitans (Wang andWalz, 2003;Wang et al., 2004) and does notneed any staining to be recognizable. As minocycline com-pletely prevented the appearance of such a cell-free space (seeResults), resulting in a cavity space of zero, quantification andstatistical analysis were not necessary.

4.5. Enzyme-linked immunosorbent assay for IL-1β

Twenty-four hours following pial vessel disruption, rats (n = 7)were perfused by ice-cold PBS and the ischemic ipsilateral andnonischemic contralateral cortices were removed and snapfrozen in liquid nitrogen and store at −80 °C until assay. Forextraction of proteins, the tissue was homogenized in 250 μl ofhomogenization buffer consisting of: 50mMTris, pH 7.3, 5mMEDTA, 1% protease inhibitor cocktail (Sigma, P8340) for every50 mg wet weight. The samples were centrifuged at 4 °C for20 min at 13,000 RPM and the supernatant was aliquoted. TheIL-1β protein concentrations in rat brain tissue homogenateswere quantified using ELISA Duoset kits (R&D system)according to the manufacturer's instructions. Initial studies

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showed that IL-1β recovery from brain tissue spiked withknown concentrations of IL-1β was in the range 85–120%.Tissue extracts (100 μl) were applied to each well for the ELISAand all the samples were duplicated. IL-1β protein concentra-tions were normalized based on the original wet weight of thebrain tissue samples.

4.6. Cell counting

The images of stained specimens were captured by a digitalcamera (Coolsnap fx) mounted on a microscope (OlympusIX71) andanalyzedby ImagePro software. For quantification, atleast 3 sections were stained and counted from each animal.

Only GSAB4-positive cells with nuclei were counted in theperi-infarct areas (the inset in Fig. 5 showed the representativefields used in quantification). Cell population density wascalculated by dividing the cell number by the brain sectionvolume. The results were corrected with the use of Abercrom-bie's technique, allowing assessment of the real density ofmicroglia (Savchenko et al., 2000). The calculation was carriedout by the formulaN = n × T / (T + D), whereN is the estimate ofthe true cell number, n is the number of nuclear segments, T isthe mean section thickness, and D corresponds to a meannuclear diameter. The number of MPO-positive cells wascounted of the entire lesion and normalized to the area ofinfarct.

4.7. Statistical analysis

The data are expressed as mean ± standard error. Student's ttest or, where appropriate, two-way ANOVA was used toidentify significant differences (P < 0.05).

Acknowledgments

Operating funds were provided by the Heart and StrokeFoundation of Saskatchewan. The authors would like tothank Dr. Michel Desautels, Dr. Ron Doucette, Dr. John Gordon,and Xiaobei Zhang for their advice and support.

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